Abstract
Treatment of MCF-7 cells with tamoxifen induced vacuole formation and cell death. Levels of the autophagy marker, microtubule-associated protein light chain 3 (LC3)-II also increased, and GFP-LC3 accumulated in and around vacuoles in MCF-7 cells exposed to tamoxifen, indicating that autophagy is involved in tamoxifen-induced changes. Live-cell confocal microscopy with FluoZin-3 staining and transmission electron microscopy with autometallographic staining revealed that labile zinc(II) ion (Zn2+) accumulated in most acidic LC3(+) autophagic vacuoles (AVs). Chelation of Zn2+ with N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) blocked the increase in phospho-Erk and LC3-II levels, and attenuated AV formation and cell death. Conversely, the addition of ZnCl2 markedly potentiated tamoxifen-induced extracellular signal-regulated kinase (Erk) activation, autophagy and cell death, indicating that Zn2+ has an important role in these events. Tamoxifen-induced death was accompanied by increased oxidative stress and lysosomal membrane permeabilization (LMP) represented as release of lysosomal cathepsins into cytosol. Treatment with the antioxidant N-acetyl-l-cysteine (NAC) blunted the increase in Zn2+ levels and reduced LC3-II conversion, cathepsin D release and cell death induced by tamoxifen. And cathepsin inhibitors attenuated cell death, indicating that LMP contributes to tamoxifen-induced cell death. Moreover, TPEN blocked tamoxifen-induced cathepsin D release and increase in oxidative stress. The present results indicate that Zn2+ contributes to tamoxifen-induced autophagic cell death via increase in oxidative stress and induction of LMP.
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Abbreviations
- 3-MA:
-
3-methyladenine
- AMG:
-
Autometallography
- ATG:
-
Autophagy-related gene
- AV:
-
Autophagic vacuole
- ER:
-
Estrogen receptor
- Erk:
-
Extracellular signal-regulated kinase
- Lamp-2:
-
Lysosomal-associated membrane protein-2
- LC3-II:
-
Microtuble-associated protein light chain 3-II
- LDH:
-
Lactate dehydrogenase
- LMP:
-
Lysosomal membrane permeabilization
- MT:
-
Metallothionein
- NAC:
-
N-acetyl-l-cysteine
- ROI:
-
Region of interest
- ROS:
-
Reactive oxygen species
- SERM:
-
Selective estrogen receptor modulator
- TPEN:
-
N,N,N’,N′-tetrakis (2-pyridylmethyl) ethylenediamine
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Acknowledgments
We thank Drs. Noboru Mizushima and Maria Colombo for generous gifts of LC3 cDNA and RFP-LC3 plasmid respectively. This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea [A084270].
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Live cell confocal microscopic images (2 minutes/frame) of MCF-7 cells transfected with LC3-GFP, 60-90 minutes after addition of 17 μM tamoxifen in HBSS. Images were obtained from z-series collections (35 z-section images of 1 μm thickness). (AVI 464 kb)
Live cell confocal microscopic images (2 minutes/frame) of a MCF-7 cell stained with FluoZin-3. Cells were treated with 17 μM tamoxifen and images were obtained from z-series collection (38 z-section images of 1 μm thickness) for 86 minutes. (AVI 170 kb)
Live cell confocal microscopic images (1 minutes/frame) of a MCF-7 cell stained with FluoZin-3. Cells were treated with 17 μM tamoxifen and images were obtained from z-series collection (33 z-section images of 1 μm thickness) for 90 minutes. (AVI 483 kb)
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Hwang, J.J., Kim, H.N., Kim, J. et al. Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line. Biometals 23, 997–1013 (2010). https://doi.org/10.1007/s10534-010-9346-9
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DOI: https://doi.org/10.1007/s10534-010-9346-9